Chimeric protein

ABSTRACT

The present invention provides a chimeric protein having the formula: Casp-Ht1-Ht2 wherein Casp is a caspase domain; Ht1 is a first heterodimerization domain; and Ht2 is a second heterodimerization domain and wherein, in the presence of a chemical inducer of dimcrization (CID), an identical pair of the chimeric proteins interact such that Ht1 from one chimeric protein heterodimerizes with Ht2 from the other chimeric protein, causing homodimerization of the two caspase domains. The invention also provides a cell comprising such a protein and its use in adoptive cell therapy.

FIELD OF THE INVENTION

The present invention relates to a chimeric protein useful in adoptivecell therapy (ACT). The chimeric protein can act as a suicide geneenabling cells expressing the chimeric protein to be deleted. Thepresent invention also provides a nucleic acid encoding such a chimericprotein, a cell comprising such a nucleic acid and therapeutic usesthereof.

BACKGROUND TO THE INVENTION Adoptive Cell Therapy

Adoptive immunotherapy is an established and evolving therapeuticapproach. In the setting of allogeneic haematopoietic stem celltransplantation (HSCT), donor lymphocyte infusions (DLI) are frequentlygiven to treat relapse of haematological malignancies. Tumourinfiltrating lymphocytes (TILs) are effective in treating metastaticmelanoma. Genetic engineering of T-cells greatly increases the scope andpotency of T-cell therapy: T-cell receptor transfer allows targeting ofintracellular cancer antigens, while chimeric antigen receptors (CAR)allow targeting of surface cancer or lineage specific antigens. Clinicalresponses have been observed with both approaches, and numerous furthertrials are underway.

Acute adverse events can occur following adoptive immunotherapy.Graft-versus-host disease (GvHD) is a common and serious complication ofDLI. Administration of engineered T-cells has also resulted in toxicity.For instance, on-target off-tumour toxicity has been reported in nativeT-cell receptor transfer studies against melanoma antigens; T-cellsre-directed to the renal cell carcinoma antigen carbonic anhydrase IX(CAIX) produced unexpected hepatotoxicity. Immune activation syndromeshave been reported after CD19 CAR therapy. Finally vector-inducedinsertional mutagenesis results in a theoretical risk oflymphoproliferative disorders. The incidence and severity of thesetoxicities is unpredictable. Further, in contrast to a therapeuticprotein or small molecules whose adverse events usually abate with thehalf-life of the therapeutic, T-cells engraft and replicate, potentiallyresulting in escalating and fulminant toxicity.

Suicide Genes

A suicide-gene is a genetically encoded mechanism which allows selectivedestruction of adoptively transferred cells, such as T-cells, in theface of unacceptable toxicity. Two suicide-genes have been tested inclinical studies: Herpes Simplex Virus thymidine kinase (HSV-TK) andinducible caspase 9 (iCasp9).

The herpes simplex virus I-derived thymidine kinase (HSV-TK) gene hasbeen used as an in vivo suicide switch in donor T-cell infusions totreat recurrent malignancy and Epstein Barr virus (EBV)lymphoproliferation after hemopoietic stem cell transplantation.However, destruction of T cells causing graft-versus-host disease wasincomplete, and the use of ganciclovir (or analogs) as a pro-drug toactivate HSV-TK precludes administration of ganciclovir as an antiviraldrug for cytomegalovirus infections. Moreover, HSV-TK-directed immuneresponses have resulted in elimination of HSV-TK-transduced cells, evenin immunosuppressed human immunodeficiency virus and bone marrowtransplant patients, compromising the persistence and hence efficacy ofthe infused T cells.

The activation mechanism behind Caspase 9 was exploited in the originaliCasp9 molecule. All that is needed for Caspase 9 to become activated,is overcoming the energic barrier for Caspase 9 to homodimerize. Thehomodimer undergoes a conformational change and the proteolytic domainof one of a pair of dimers becomes active. Physiologically, this occursby binding of the CARD domain of Caspase 9 to APAF-1. In iCasp9, theAPAF-1 domain is replaced with a modified FKBP12 which has been mutatedto selectively bind a chemical inducer of dimerization (CID). Presenceof the CID results in homodimerization and activation. iCasp9 is basedon a modified human caspase 9 fused to a human FK506 binding protein(FKBP) (Straathof et al (2005) Blood 105:4247-4254). It enablesconditional dimerization in the presence of a small molecule CID, knownas AP1903. AP1903 is an experimental drug and is considered biologicallyinert since it does not interact with wild-type FKBP12. However clinicalexperience with this agent is limited to a very small number of patients(Di Stasi, A. et al. (2011) N. Engl. J. Med. 365, 1673-1683; andluliucci, J. D. et al. (2001) J. Clin. Pharmacol. 41, 870-879). AP1903is also a relatively large and polar molecule and unlikely to cross theblood-brain barrier.

In an alternative approach, executioner caspases can be activated bysmall molecules using a complex strategy which involves introduction oftobacco etch virus (TeV) proteolysis sites into Caspase 3 or 6 or 7 andco-expression with a split TEV protease which is recombined in thepresence of rapamycin (Morgan et al (2014) Methods Enzymol.544:179-213). This is an unsatisfactory strategy for a clinically usefulsuicide switch for a number of reasons: firstly three separate proteinsare required which is highly complex: the modified caspase, and the twocomponents of the split TeV protease respectively; secondly, TeVcomponents are xenogeneic and likely immunogenic; finally, this strategyonly activates protease sensitive caspase molecules which are downstreamand less sensitive than apical caspases.

A suicide gene based on CID activation of FAS has been described (Amaraet al (1999) Hum. Gene Ther. 10, 2651-2655). This also depends on thisCID for activation, and since it does not directly activate theapoptosis cascade, escape (through FAS resistance) is possible.

A homodimerization system based on a standard pharmaceutical whichreplaces the need for an experimental CID would be an attractivealternative. However, no homodimerizing small molecule pharmaceuticalsare available.

Other suicide genes have been proposed for instance full-length CD20when expressed on a T-cell can render T-cells susceptible to lysis bythe therapeutic anti-CD20 antibody Rituximab (Introna, M. et al. (2000)Hum. Gene Ther. 11, 611-620). Further suicide genes have also beendescribed on this theme of antibody recognition, for example: RQR8renders T-cells susceptible to CD20 but is more compact than thefull-length CD20 molecule (Philip, B. et al. (2014) Blooddoi:10.1182/blood-2014-01-545020); a truncated version of EGFR (huEGFRt)renders cells susceptible to lysis by anti-EGFR mAbs (Wang, X. et al.(2011) Blood 118, 1255-1263); and a myc epitope tag expressed on a cellsurface leaves cells susceptible to lysis with an anti-myc antibody(Kieback et al (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 623-628). Amajor limitation of these antibody dependent approaches is theirdependence on bioavailability of a therapeutic antibody at high localconcentrations to act. It is known for instance that lytic antibodiesare not particularly effective against bulky disease and a limitation ofantibody based suicide genes is that cells resident where high antibodyconcentrations are not reached would escape. Further, in certainsituations: for instance a severe macrophage activation syndrome orcytokine storm induced by a CAR T-cells; the additional immuneactivation induced by a monoclonal antibody may be deleterious to theclinical situation activation of the suicide gene is trying to treat.

There is thus a need for an alternative suicide gene which is notassociated with the disadvantages mentioned above.

DESCRIPTION OF THE FIGURES

FIG. 1—Cartoons showing different approaches to RapCasp9. (a) Doubleconstruct where two molecules are expressed separately. Each moleculehas the catalytic domain of Casp9 fused with either FKBP12 or FRBrespectively. (b) Single construct where FKBP12 and FRB are directlyfused together and then fused to the catalytic domain of Casp9 by aflexible linker. Self heterodimerization should not be possible in thisorientation. (c) Single construct where the catalytic domain of Caspase9 is flanked by FRB and FKBP12. Here, self heterodimerization may occurso this iteration is not expected to function well. (d) Double constructwhere the catalytic domain of Caspase 9 is fused to FKBP12 and aseparate small protein which is a fusion of two copies of FRB isco-expressed.

FIG. 2—Demonstration that it is possible to activate Caspase 9 with aheterodimerizer. T-cells were either transduced with eGFP alone (FIG. 2a), or co-transduced with FKBP12-dCasp9 (co-expressing eGFP) andFRB-dCasp9 (co-expressing eBFP2) (FIG. 2b ). T-cells were intentionallyonly partially transduced so that the non-transduced T-cells would actas internal controls. T-cells were then exposed to decreasingconcentrations of Rapamycin. After 48 hours, cells were stained withAnnexin-V and 7AAD and analysed by flow cytometry looking at theproportion of live cells which were expressing fluorescent proteins.T-cells expressing both eGFP and eBFP2 were very effectively deletedeven in the presence of the lowest concentration of Rapamycin.

FIG. 3—Function of RapCasp9 variants. T-cells were transduced with (a)eGFP alone; (b) double transduced with FKBP12-Casp9 and FRB-Casp9co-expressed with eGFP and eBFP2 respectively; (c) transduced withFRB-FKBP12-Casp9 and (d) transduced with FRB-Casp9-FKBP12 and (e)FBP12-Casp9-2A-FRB-FRBw. Only a proportion of cells were transduced, thenegative cells acted as an internal negative control. T-cells wereexposed for 48 hours to 2.5nM Rapamycin. T-cells were then stained withAnnexin-V and 7AAD and analysed by flow-cytometry. eGFP vs eBFP2 isshown on live cells as determined by Annexin-V and 7AAD staining.

FIG. 4—Rapamycin and rapalogs. A) Rapamycin; B) C-20-methyllyrlrapamycin(MaRap); C) C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap); D)C16-(S)-3-mehylindolerapamycin (C16-iRap); and E)C16-(S)-7-methylindolerapamycin (AP21976/C16-AiRap).

FIG. 5—Summary of the constructs tested in Example 3.

FIG. 6—Summary of gating strategy for Example 3.

FIGS. 7, 8 and 9—Study showing the killing of Jurkat cells transfectedwith the constructs shown in FIG. 5 after incubation with variousconcentrations of rapamycin.

FIG. 10—Graph to summarise the FACS data shown in FIGS. 7, 8 and 9.

FIG. 11—Graph comparing Jurkat cell killing in the presence of rapamycinvs temsirolimus.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have developed a new suicide gene, which dimerizesin the presence of a chemical inducer of dimerization (CID) such asrapamycin or a rapamycin analogue.

Rapamycin and rapamycin analogues induce heterodimerisation bygenerating an interface between the FRB domain of mTOR and FKBP12. Thisassociation results in FKBP12 blocking access to the mTOR active siteinhibiting its function. While mTOR is a very large protein, the precisesmall segment of mTOR required for interaction with Rapamycin is knownand can be used.

The present inventors have shown that it is possible to use theheterodimerization mediated by rapamycin to induce homodimerization of acaspase. In particular, they have surprisingly shown that it is possibleto create a multi-domain molecule, which includes (i) the FRB domain ofmTOR; (ii) FKBP12; and (iii) a caspase, and use heterodimerizationbetween the FRB domain of one copy of the molecule and the FKB12 domainof another copy of the molecule to cause homodimerization of the caspasedomains.

Thus in a first embodiment of the first aspect of the invention, thepresent invention provides a chimeric protein having the formula:

Ht1-Ht2-Casp

whereinCasp is a caspase domain;Ht1 is a first heterodimerization domain; andHt2 is a second heterodimerization domainand wherein, in the presence of a chemical inducer of dimerization(CID), an identical pair of the chimeric proteins interact such that Ht1from one chimeric protein heterodimerizes with Ht2 from the otherchimeric protein, causing homodimerization of the two caspase domains.

The configuration is such that Ht1 does not heterodimerize to anysignificant extent with Ht2 within the same chimeric protein.

The caspase domain may comprise an initiator caspase selected from thefollowing group: caspase-8, caspase-9 and caspase-10, or an executionercaspase selected from caspase-3 and caspase-7.

In the multi-domain protein of this first embodiment of the first aspectof the invention one heterodimerization domain may comprise anFK506-binding protein (FKBP) and the other heterodimerization domain maycomprise an FRB domain of mTOR.

For this heterodimerization domain combination, a suitable CID israpamycin or a rapamycin analog.

In a second embodiment of the first aspect of the invention there isprovided a chimeric protein which comprises a caspase domain and aheterodimerization domain which comprises an FK506-binding protein(FKBP12), and a chimeric protein which comprises a caspase domain and aheterodimerization domain which comprises an FRB domain of mTOR.

In a third embodiment of this aspect of the invention there are providedtwo proteins:

-   -   Ht1-Casp and Ht2-Ht2    -   wherein Ht1-Casp is a chimeric protein comprising a caspase        domain (Casp) and a first heterodimerization domain (Ht1); and        Ht2-Ht2 is an interfacing protein comprising two or more second        heterodimerization domains (Ht2); and        wherein, in the presence of a chemical inducer of dimerization        (CID), a pair of the chimeric proteins Ht1-Casp9 interact such        that Ht1 from each chimeric protein heterodimerizes with an Ht2        domain from the interfacing protein, causing homodimerization of        the two caspase domains.

In a fourth embodiment of this aspect of the invention there is provideda chimeric protein having the formula:

Ht1-Casp-Ht2

-   -   wherein    -   Casp is a caspase domain;    -   Ht1 is a first heterodimerization domain; and    -   Ht2 is a second heterodimerization domain        and wherein, in the presence of a chemical inducer of        dimerization (CID), an identical pair of the chimeric proteins        interact such that Ht1 from one chimeric protein heterodimerizes        with Ht2 from the other chimeric protein, causing        homodimerization of the two caspase domains.

With this fourth embodiment of the first aspect of the invention, whereone heterodimerization domain comprises an FK506-binding protein (FKBP)and the other heterodimerization domain comprises an FRB domain of mTORand the CID is rapamycin or a derivative thereof, then concentrations ofless that 5 nm, for example 1-3 nm or about 1 nm may be used in order tocause homodimerisation of the two caspase domains.

The chimeric protein may comprise a caspase domain fused to FKBP12 andis the interfacing protein may be a fusion of two or more FRB domains.These two or more FRB domains act as an interface, brining twoFKBP12-Casp domains together. In a second aspect, the present inventionprovides a nucleic acid sequence which encodes a chimeric proteinaccording to the first aspect of the invention.

The nucleic acid may be in the form of a nucleic acid construct, whichcomprises a plurality of nucleic acid sequences. For example, theconstruct may comprise one or more nucleic acid sequence(s) according tothe second aspect of the invention and a nucleic acid sequence encodinga T-cell receptor (TCR) or chimeric antigen receptor (CAR).

The nucleic acid construct may comprise:

i) a first nucleic acid sequence encoding a chimeric protein whichcomprises a caspase domain and a heterodimerization domain whichcomprises an FK506-binding protein (FKBP);ii) a second nucleic acid sequence encoding a chimeric protein whichcomprises a caspase domain and a heterodimerization domain whichcomprises an FRB domain of mTOR.

There is also provided a nucleic acid construct having the structure:

Ht1-Casp-coexpr-Ht2-Ht2

-   -   wherein:    -   Casp is a nucleic acid sequence encoding a caspase domain;    -   Ht1 is a nucleic acid sequence encoding a first        heterodimerization domain;    -   Ht2 is a nucleic acid sequence encoding a second        heterodimerization domain; and    -   coexpr is a nucleic acid sequence allowing co-expression of        Ht1-Casp and Ht2-Ht2,    -   wherein expression of the nucleic acid construct results in the        production of a chimeric protein Ht1-Casp and an interfacing        protein Ht2-Ht2 and wherein, in the presence of a chemical        inducer of dimerization (CID), a pair of the chimeric proteins        Ht1-Casp interact such that Ht1 from each chimeric protein        heterodimerizes with an Ht2 domain from the interfacing protein,        causing homodimerization of the two caspase domains.

Ht1 may comprise an FK506-binding protein (FKBP) and Ht2 may comprise anFRB domain of mTOR.

The nucleic acid construct may also comprise a nucleic acid sequenceencoding a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

In a third aspect, the present invention provides a vector whichcomprises a nucleic acid sequence or a nucleic acid construct accordingto the second aspect of the invention.

The vector which may also comprise a nucleotide of interest, such as anucleotide sequence encoding a chimeric antigen receptor or a T-cellreceptor, such that when the vector is used to transduce a target cell,the target cell co-expresses a chimeric protein according to the firstaspect of the invention and a chimeric antigen receptor or T-cellreceptor.

In a fourth aspect the present invention provides a cell which expressesa chimeric protein according to the first aspect of the invention.

The cell may comprise:

i) a first chimeric protein which comprises a caspase domain and aheterodimerization domain which comprises an FK506-binding protein(FKBP); andii) a second chimeric protein which comprises a caspase domain and aheterodimerization domain which comprises an FRB domain of mTOR.

There is also provided a cell which expresses two proteins:

-   -   Ht1-Casp and Ht2-Ht2    -   wherein Ht1-Casp is a chimeric protein comprising a caspase        domain (Casp) and a first heterodimerization domain (Ht1); and        Ht2-Ht2 is an interfacing protein comprising two second        heterodimerization domains (Ht2); and    -   wherein, in the presence of a chemical inducer of dimerization        (CID), a pair of the chimeric proteins Ht1-Casp9 interact such        that Ht1 from each chimeric protein heterodimerizes with an Ht2        domain from the interfacing protein, causing homodimerization of        the two caspase domains.

The cell may comprise a nucleic acid sequence or construct according tothe second aspect of the invention.

The cell may, for example, be a haematopoietic stem cell, a lymphocyteor a T cell.

There is also provided a method for making a cell according to thefourth aspect of the invention which comprises the step of transducingor transfecting a cell with a vector according to the third aspect ofthe invention.

There is also provided a method for deleting a cell according to thefourth aspect of the invention, which comprises the step of exposing thecells to a chemical inducer of dimerization (CID).

The CID may be rapamycin or a rapamycin analog.

There is also provided a method for preventing or treating a disease ina subject, which comprises the step of administering a cell according tothe fourth aspect of the invention to the subject.

The method may comprise the following steps:

-   -   (i) transducing or transfecting a sample of cells isolated from        a subject with a vector according to the second aspect of the        invention, and    -   (ii) administering the transduced/transfected cells to a        patient.

The method may be for treating cancer.

There is also provided a method for preventing and/or treating anpathological immune reaction in a subject caused by administration of acell according to the fourth aspect of the invention to the subject,which comprises the step of administering rapamycin or a rapamycinanalog to the subject.

The pathological immune reaction may be selected from the followinggroup: graft-versus-host disease; on-target, off-tumour toxicity; immuneactivation syndrome; and lymphoproliferative disorders.

The method for treating or prevention a disease in a subject maycomprise the following steps:

(i) administering a cell according to the fourth aspect of the inventionto the subject;(ii) monitoring the subject for the development of a pathological immunereaction; and(iii) administering rapamycin or a rapamycin analog to the subject ifthe subject shows signs of developing or having developed a pathologicalimmune reaction.

There is also provided a cell according to the fourth aspect of theinvention for use in haematopoietic stem cell transplantation,lymphocyte infusion or adoptive cell transfer.

There is also provided rapamycin or a rapamycin analog for use inpreventing or treating a pathological immune reaction caused byadministration of a cell according to the fourth aspect of the inventionto a subject.

Thus the present invention provides a suicide gene which allows theselective destruction of adoptively infused cells in the face ofunacceptable toxicity, and which is activated by rapamycin and/or itsanalogues.

Rapamycin is standard pharmaceutical with well understood properties,excellent bioavailability and volume of distribution and which is widelyavailable. Rapamycin also does not aggravate the condition beingtreated, in fact, as it is an immunosuppressant it is likely to have abeneficial effect on unwanted toxicity as well as its suicide genefunction.

DETAILED DESCRIPTION Chimeric Protein

The present invention relates to a chimeric protein which acts as asuicide gene. Cells expressing the chimeric protein may be deleted invivo or in vitro by administration of a chemical inducer of dimerization(CID) such as rapamycin or a rapamycin analogue.

The chimeric protein may have the formula:

Ht1-Ht2-Casp

in whichCasp is a caspase domain;Ht1 is a first heterodimerization domain; andHt2 is a second heterodimerization domain.

The chimeric protein may have the formula:

Ht1-Ht2-L-Casp

in which Casp, Ht1 and Ht2 are as defined above and L is an optionallinker.

The configuration should be such that Ht1 does not significantlyheterodimerize with Ht2 within the same chimeric protein molecule, butwhen two chimeric proteins come together in the presence of a chemicalinducer of dimerization (CID) Ht1 from one chimeric proteinheterodimerizes with Ht2 from the other chimeric protein, causinghomodimerization of the two caspase domains.

The configuration is such that Ht1 does not heterodimerize to anysignificant extent with Ht2 within the same chimeric protein. Forexample, in a cell expressing a chimeric protein according to thisembodiment of the first aspect of the invention, the presence of the CIDshould cause a greater proportion of dimerization between two chimericproteins, than heterodimerization within the same chimeric protein. Theamount of chimeric proteins which are heterodimerized within the samemolecule in a cell or cell population, or in solution, may be less than50%, 40%, 30%, 20%, 10%, 5% or 1% of the amount of chimeric proteinswhich are heterdomerized with a separate chimeric protein molecule, inthe presence of the CID.

The chimeric protein may comprise the sequence shown as SEQ ID No. 1.

(FRB-FKBP12-L3-dCasp9 ) SEQ ID No. 1<-----------------------FRB---------------------------------MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR-----------------FRB-------------------><L1-><--FKBP12------DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEGVQVETISPGDGR------------------FKBP12------------------------------------TFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK---------------------------------><------L3------><--dCasp9-LTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGSGGGGSGVDGFGDVGA------------------------dCasp9------------------------------LESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMV------------------------dCasp9------------------------------EVKGDLTAKKMVLALLELAQQDHCALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVE------------------------dCasp9------------------------------KIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQE------------------------dCasp9------------------------------GLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQ--------------dCasp9---------------->SLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS

In the above sequence “FKBP12” refers to the sequence of FKBP12;“dCasp9” refers to the catalytic domain of Casp9; “L1” is a one repeatlinker; “FMD-2A” is a Foot and mouth disease 2A like peptide ERAV; “FRB”is the FRB domain of mTOR; “L3” is a two repeat linker; and “FRBw” iscodon wobbled FRB

In a second embodiment, the invention provides a “two-molecule” suicidegene system, in which the CID is rapamycin or a rapamycin analogue.

Thus, the present invention also provides i) a chimeric protein whichcomprises a caspase domain and a heterodimerization domain whichcomprises an FK506-binding protein (FKBP12); and ii) a chimeric proteinwhich comprises a caspase domain and a heterodimerization domain whichcomprises an FRB domain of mTOR.

When a cell, such as a T-cell, expresses both these chimeric proteins,the presence of rapamycin or a rapamycin analogue causes theFKBP-comprising domain or i) to heterodimerise with the FRB-comprisingdomain or ii), thus causing homodimerization of the caspase domains fromi) and ii).

In this embodiment of the invention, the chimeric protein may comprisethe sequence shown as SEQ ID No. 2 or 3.

(FKBP12-dCasp9) SEQ ID No. 2<-------------------FKBP12----------------------------------MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKEDSSRDRNKPFKFMLGKQEVIR--------------------------FKBP12-----------------><L1-><----GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF-----------------------dCasp9-------------------------------GDVGALESLRGNADLAYILSMEPCGHCLIINNVNECRESGLRTRTGSNIDCEKLRRRESS-----------------------dCasp9-------------------------------LHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGC-----------------------dCasp9-------------------------------PVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDA-----------------------dCasp9-------------------------------TPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAH-----------------------dCasp9------------>SEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS (FRB-dCasp9) SEQ ID No. 3---------------------------FRB----------------------------->MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERCPQTLKETSFNQAYGR                      FRB              ><L1 ><    dCasp9DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSGVDGFGDVGALESLR-----------------------dCasp9-------------------------------GNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGD-----------------------dCasp9-------------------------------LTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNI-----------------------dCasp9-------------------------------FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTF-----------------------dCasp9-------------------------------DQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLR-------------dCasp9------------> VANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS

In a third embodiment, the invention provides an alternative “twomolecule” approach, with a smaller footprint than the second embodiment.Here, Ht1 is fused with Caspase, and a second molecule comprises ofHt2-Ht2 fusion is co-expressed. In the prescence of CID, Ht2-Ht2 bringstogether two Ht1-Casp molecules. In practise, this can be implemented byco-expressing FKBP12-Casp9 with FRB-FRB and activating with Rapamycin.Conveniently, these components can be co-expressed with a foot-and-mouthdisease 2A like peptide. The second Ht2 (for example FRB) encodingsequence may be codon wobbled to prevent recombination.

(FKBP12-dCasp9-2A-FRB-FRBw) SEQ ID No. 4 <                   FKBP12MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIR--------------------------FKBP12-----------------><L1-><----GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF-----------------------dCasp9-------------------------------GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSS-----------------------dCasp9-------------------------------LHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGC-----------------------dCasp9-------------------------------PVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDA-----------------------dCasp9-------------------------------TPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAH------------dCasp9-----------------------><----FMD-2A-------SEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSASQCTNYALLKLAGDVESNP-><--------------------FRB----------------------------------GPGVQVETISPGDGRTFPKRCQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG---------------FRB------------------------------><----L2--->WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGS<---------------------------FRBw----------------------------MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIR-----------------------FRBw----------------------->GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLES

In the above sequence: “FKBP12” refers to FKBP12; “dCasp9” is thecatalytic domain of Casp9; “L1” is a one repeat linker; “FMD-2A” is aFoot and mouth disease 2A like peptide ERAV; “FRB” is the FRB domain ofmTOR; “L2” is a two repeat linker; and “FRBw” is codon wobbled FRB.

Caspase

Caspases, or cysteine-aspartic proteases or cysteine-dependentaspartate-directed proteases are a family of cysteine proteases thatplay essential roles in apoptosis.

Twelve caspases have been identified in humans. There are two types ofapoptotic caspases: initiator caspases and executioner caspases.Initiator caspases, such as caspase-2, caspase-8, caspase-9, andcaspase-10, cleave inactive pro-forms of effector caspases, therebyactivating them. Executioner caspases, such as caspase-3, caspase-6 andcaspase-7, then cleave other protein substrates within the cell, totrigger the apoptotic process.

The caspase domain of the chimeric protein of the first aspect of thepresent invention may comprise an initiator caspase selected fromcaspase-2; caspase-8, caspase-9 and caspase-10; or an executionercaspase selected from caspase-3, caspase-6 and caspase-7.

In particular, the caspase domain of the chimeric protein of the firstaspect of the present invention may comprise caspase-9. Caspase 9 is thekey initiator caspase so its activation is a very sensitive trigger forapoptosis induction. Furthermore, homodimerization is all that isrequired for activation, rather than homodimerization and proteolyticcleavage.

Full length caspase-9 has the sequence shown as SEQ ID No. 5.

(Caspase-9) SEQ ID No. 5   1MDEADRRLLR RCRLRLVEEL QVDQLWDALL SSELFRPHMI EDIQRAGSGS RRDQARQLII  61DLETRGSQAL PLFISCLEDT GQDMLASFLR TNRQAAKLSK PTLENLTPVV LRPEIRKPEV 121LRPETPRPVD IGSGGFGDVG ALESLRGNAD LAYILSMEPC GHCLIINNVN FCRESGLRTR 181TGSNIDCEKL RRRFSSPHFM VEVKGDLTAK KMVLALLELA QQDHGALDCC VVVILSHGCQ 241ASHLQFPGAV YGTDGCPVSV EKIVNIFNGT SCPSLGGKPK LFFIQACGGE QKDHGFEVAS 301TSPEDESPGS NPEPDATPFQ EGLRTFDQLD AISSLPTPSD IFVSYSTFPG FVSWRDPKSG 361SWYVETLDDI FEQWAHSEDL QSLLLRVANA VSVKGIYKQM PGCFNFLRKK LFFKTS

Caspase-9 may be truncated, for example to remove the caspaserecruitment domain. Truncated Caspase-9 is shown as SEQ ID No. 6

(truncated Caspase-9, lacking the CARD domain) SEQ ID No. 6GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

The chimeric protein of the first aspect of the invention may compriseSEQ ID No. 5 or SEQ ID No. 6 or a fragment or a variant thereof whichretains the capacity to homodimerize and thus trigger apoptosis.

A variant caspase-9 sequence may have at least 80%, 85%, 90%, 95%, 98%or 99% sequence identity to SEQ ID No. 5 or 6.

The percentage identity between two polypeptide sequences may be readilydetermined by programs such as BLAST which is freely available athttp://blast.ncbi.nlm.nih.gov.

In vivo, the protease caspase 9 is the central participant in amulti-component pathway known as the apoptosome, which controls celldeletion during embryogenesis, and physiological responses that triggercell death as well as lethal cellular insults such as ionizing radiationor chemotherapeutic drugs. The function of caspase 9 is to generate theactive forms of caspases 3 and 7 by limited proteolysis, and therebytransmit the apoptotic signal to the execution phase. However, caspase 9is unusual among its close relatives in that proteolysis between thelarge and small subunit does not convert the latent zymogen to thecatalytic form. In fact, it is homodimerization which is required foractivation.

Heterodimerization Domains

The macrolides rapamycin and FK506 act by inducing theheterodimerization of cellular proteins. Each drug binds with a highaffinity to the FKBP12 protein, creating a drug-protein complex thatsubsequently binds and inactivates mTOR/FRAP and calcineurin,respectively. The FKBP-rapamycin binding (FRB) domain of mTOR has beendefined and applied as an isolated 89 amino acid protein moiety that canbe fused to a protein of interest. Rapamycin can then induce theapproximation of FRB fusions to FKBP12 or proteins fused with FKBP 12.

In the context of the present invention one of the heterodimerizationdomains (Ht1 or Ht2) may be or comprise FRB, or a variant thereof andthe other heterodimerization domain (Ht2 or Ht1) may be or compriseFKBP12 or a variant thereof.

Rapamycin has several properties of an ideal dimerizer: it has a highaffinity (KD<1 nM) for FRB when bound to FKBP12, and is highly specificfor the FRB domain of mTOR. Rapamycin is an effective therapeuticimmunosuppressant with a favourable pharmacokinetic and pharmacodynamicsprofile in mammals. Pharmacological analogues of Rapamycin withdifferent pharmacokinetic and dynamic properties such as Everolimus,Temsirolimus and Deforolimus (Benjamin et al, Nature Reviews, DrugDiscovery, 2011) may also be used according to the clinical setting.

In order to prevent rapamycin binding and inactivating endogenous mTOR,the surface of rapamycin which contacts FRB may be modified.Compensatory mutation of the FRB domain to form a burface thataccommodates the “bumped” rapamycin restores dimerizing interactionsonly with the FRB mutant and not to the endogenous mTOR protein.

Bayle et al. (Chem Bio; 2006; 13; 99-107) describes various rapamycinanalogs, or “rapalogs” and their corresponding modified FRB bindingdomains. For example,

Bayle et al. (2006) describes the rapalogs: C-20-methyllyrlrapamycin(MaRap), C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap) andC16-(S)-7-methylindolerapamycin (AP21976/C16-AiRap), as shown in FIG. 3,in combination with the respective complementary binding domains foreach. Other rapamycins/rapalogs include sirolimus and tacrolimus.

The heterodimerization domains of the chimeric protein may be orcomprise one the sequences shown as SEQ ID NO: 7 to SEQ ID NO: 11, or avariant thereof.

-FKBP12 domain SEQ ID No 7MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVF DVELLKLE-wild-type FRB segment of mTOR SEQ ID No 8MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKLES-FRB with T to L substitution at 2098 which allows binding to AP21967SEQ ID No 9 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLES-FRB segment of mTOR with T to H substitution at2098 and to W at F at residue 2101 of the fullmTOR which binds Rapamycin with reduced affinity to wild typeSEQ ID No 10 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLHQAFDLYYHVFRRISKLES-FRB segment of mTOR with K to P substitution atresidue 2095 of the full mTOR which bindsRapamycin with reduced affinity SEQ ID No 11MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVPDLTQAWDLYYHVFRRISKLES

Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99%sequence identity to SEQ ID No. 7 to 11, provided that the sequencesprovide an effective dimerization system. That is, provided that thesequences facilitate sufficient co-localisation of the two chimericproteins to allow homodimerization of the two caspase domains.

The “wild-type” FRB domain shown as SEQ ID No. 8 comprises amino acids2025-2114 of human mTOR. Using the amino acid numbering system of humanmTOR, the FRB sequence of the chimeric protein of the invention maycomprise an amino acid substitution at one of more of the followingpositions: 2095, 2098, 2101.

The variant FRB used in the chimeric protein of the invention maycomprise one of the following amino acids at positions 2095, 2098 and2101:

2095: K, P, T or A 2098: T, L, H or F 2101: W or F

Bayle et al (as above) describe the following FRB variants, annotatedaccording to the amino acids at positions 2095, 2098 and 2101 (see Table1): KTW, PLF, KLW, PLW, TLW, ALW, PTF, ATF, TTF, KLF, PLF, TLF, ALF,KTF, KHF, KFF, KLF. These variants are capable of binding rapamycin andrapalogs to varying extents, as shown in Table 1 and FIG. 5A of Bayle etal. The chimeric protein of the invention may comprise one of these FRBvariants.

Linker

A linker may be included to spatially separate the caspase domain andthe heterodimerization domain(s).

In the first embodiment of the first aspect of the present invention,the chimeric protein comprises two heterodimerization domains which areheld in a configuration such that they cannot heterodimerize with eachother in the presence of the CID in a single molecule, but Ht1 on onemolecule can heterodimerise with Ht2 on another chimeric molecule havingthe same heterodimerization domains (FIG. 1B). In a design where Ht1 andHt2 flank the Caspase domain (Ht1-Casp-Ht2), activation was inferior todesigns where Ht1 and Ht2 were linked together, indicating theimportance of preventing non-productive binding of Ht1 and Ht2 from asingle molecule to a single CID.

In this embodiment, the linker (L1) should provide sufficientflexibility so that the catalytic domains can homodimerize, but not somuch flexibility that the energic barrier to homodimerization is notovercome (FIG. 1). For example, the linker may be less than 15, lessthan 10 or between 5-15 or 5-10 amino acids in length.

In the second embodiment of the first aspect of the present invention,the chimeric protein comprises a single heterodimerization domain, whichis capable of heterodimerization with a complementary heterodimerizationdomain on a second chimeric protein in the presence of a CID.

In an alternative configuration, the two heterodimerisation domains maybe provided on a signle molecule with a long linker (L2), providing aconstruct having the formula:

Ht1-Casp1-L2-Ht2-Casp2

The HT and Casp domains may be in either order on each side of thelinker.

In this embodiment, the linker L2 may confer sufficient flexibility sothe first heterodimerization domain can heterodimerize with the secondheterodimerization domain; and so that the caspase domain in the part ofthe molecule corresponding to the ‘first chimeric protein’ canhomodimerize with the caspase domain in the part of the moleculecorresponding to the ‘second chimeric protein’.

In the third embodiment of the first aspect of the invention, Casp isfused to a single heterodimerization domain, but a second molecule whichis a fusion of two or more copies of the other heterodimerizationdomain. The two molecules may be co-expressed. In this case, the secondmolecule acts as an interface bringing two or more Casp domains togetherin the presence of CID. In this case, the two or more copies ofheterodimerization domains must be fused in such a way to allowapproximation of the Casp9 domains sufficiently to activate them.

The interfacing protein may be multimeric, comprising more than two Ht2domains.

For example, it is possible to combine a plurality of Ht2 domains in asingle interfacing protein using a multimerising linker such as a coiledcoil domain.

In this embodiment the interfacing protein may have the formulaHt2-L2-Ht2, or Ht2-L2 in which L2 is a coiled-coil domain.

A coiled coil is a structural motif in which two to seven alpha-helicesare wrapped together like the strands of a rope. The structure of coiledcoil domains is well known in the art. For example as described by Lupas& Gruber (Advances in Protein Chemistry; 2007; 70; 37-38).

Coiled coils usually contain a repeated pattern, hxxhcxc, of hydrophobic(h) and charged (c) amino-acid residues, referred to as a heptad repeat.The positions in the heptad repeat are usually labeled abcdefg, where aand d are the hydrophobic positions, often being occupied by isoleucine,leucine, or valine. Folding a sequence with this repeating pattern intoan alpha-helical secondary structure causes the hydrophobic residues tobe presented as a ‘stripe’ that coils gently around the helix inleft-handed fashion, forming an amphipathic structure. The mostfavourable way for two such helices to arrange themselves in thecytoplasm is to wrap the hydrophobic strands against each othersandwiched between the hydrophilic amino acids. Thus, it is the burialof hydrophobic surfaces that provides the thermodynamic driving forcefor the oligomerization. The packing in a coiled-coil interface isexceptionally tight, with almost complete van der Waals contact betweenthe side-chains of the a and d residues.

Examples of proteins which contain a coiled coil domain include, but arenot limited to, kinesin motor protein, hepatitis D delta antigen,archaeal box C/D sRNP core protein, cartilage-oligomeric matrix protein(COMP), mannose-binding protein A, coiled-coil serine-rich protein 1,polypeptide release factor 2, SNAP-25, SNARE, Lac repressor orapolipoprotein E.

Chemical Inducer of Dimerization (CID)

The chemical inducer of dimerization (CID) may be any molecule whichinduces heterodimerization between Ht1 and Ht2 on separate chimericmolecules having the same Ht1 and Ht2 domains.

The CID may be rapamycin or a rapamycin analog (“rapalogs”) which haveimproved or differing pharmadynamic or pharmacokinetic properties torapamycin but have the same broad mechanism of action. The CID may be analtered rapamycin with engineered specificity for complementary FKBP12or FRB—for example as shown in FIG. 4. Bayle et al (2006, as above)describes various rapalogs functionalised at C16 and/or C20.

Examples of such rapalogs in the first category include Sirolimus,Everolimus, Temsirolimus and Deforolimus. Examples of rapalogs in thesecond category include C-20-methyllyrlrapamycin (MaRap);C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap);C16-(S)-3-mehylindolerapamycin (C16-iRap); andC16-(S)-7-methylindolerapamycin (AP21976/C16-AiRap).

Homodimerisation of the caspase domains in the presence of CID mayresult in caspase activation which is 2, 5, 10, 50, 100, 1,000 or10,000-fold higher than the caspase activity which occurs in the absenceof CID.

Rapamycin is a potent immunsuppressive agent. Analogues of rapamycin(rapalogues) are in every day clinical use. Modern rapalogues haveexcellent bioavailability and volumes of distribution. Although they arepotent immunsuppressive agents, a short dose (to activate a suicidegene) should have minimal side-effects. Further, unlike administrationof a mAb, the pharmacological effects of rapamycin and analogues maywell be advantageous in clinical scenarios where suicide genes requireactivation, such as off-tumour toxicity or immune hyperactivationsyndromes.

Nucleic Acid Sequences

The second aspect of the invention provides a nucleic acid sequencewhich encodes a chimeric protein according to the invention.

As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleicacid” are intended to be synonymous with each other.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acids can encode the same polypeptide as aresult of the degeneracy of the genetic code. In addition, it is to beunderstood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described here to reflect the codon usageof any particular host organism in which the polypeptides are to beexpressed.

Nucleic acids according to the second aspect of the invention maycomprise DNA or RNA. They may be single-stranded or double-stranded.They may also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of theuse as described herein, it is to be understood that the polynucleotidesmay be modified by any method available in the art. Such modificationsmay be carried out in order to enhance the in vivo activity or life spanof polynucleotides of interest.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence.

In the first embodiment of this aspect of the invention there isprovided a nucleic acid which encodes a chimeric protein having theformula:

Ht1-Ht2-L-Casp

whereinHt1 is a first heterodimerization domain; andHt2 is a second heterodimerization domain.L is an optional linker;Casp is a caspase domain;

The nucleic acid sequence may encode the chimeric protein sequence shownas SEQ ID No. 1 or a variant thereof.

For example the nucleotide sequence may comprise the sequence shown asSEQ ID No. 12

(FRB-FKBP12-L3-Casp9) SEQ ID No. 12ATGGCTTCTAGAATCCTCTGGCATGAGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCTCCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCTCAAAGCTCGAGTATAGCGGCGGCGGCAGCCTGGAGGGCGTGCAGGTGGAGACCATCAGCCCAGGCGACGGCAGAACCTTCCCCAAGAGAGGCCAGACCTGCGTGGTGCACTATACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCCGCGACCGCAATAAGCCCTTCAAGTTCATGCTGGGCAAGCAGGAGGTGATCAGAGGCTGGGAGGAGGGCGTGGCCCAGATGAGCGTGGGCCAGAGAGCCAAGCTGACCATCAGCCCCGACTACGCCTATGGCGCCACCGGCCACCCCGGCATCATCCCACCCCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGAGTCCGGCGGAGGCGGGTCTGGAGGAGGCGGCAGCGGCGGCGGCGGGTCAGGCGTGGATGGCTTCGGCGACGTGGGAGCCCTGGAGAGCCTGAGAGGCAACGCCGATCTGGCCTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCCGGGAGAGCGGCCTGCGGACCCGGACCGGCAGCAACATCGACTGCGAGAAGCTGAGGAGGCGCTTCTCCTCCCTGCACTTTATGGTGGAGGTGAAAGGCGATCTGACTGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCCCAGCACCACCACCCACCCCTCCATTCCTCTCTCCTCCTCATCCTGTCCCACCGCTCCCACCCCAGCCACCTGCAGTTCCCCGGAGCCGTGTACGGCACCGACGGCTGTCCCGTGTCCGTGGAGAAGATCGTGAACATCTTCAACGGCACCTCCTGCCCCTCCCTGGGCGGCAAGCCCAAGCTGTTCTTTATCCAGGCCTGTGGCGGCGAGCAGAAGGACCACGGCTTTGAGGTGGCCAGCACCTCCCCCGAGGACGAGAGCCCAGGCAGCAACCCCGAGCCCGACGCCACCCCCTTCCAGGAGGGCCTGCGCACCTTCGACCAGCTGGACGCCATCAGCAGCCTGCCCACCCCCAGCGACATCTTCGTGAGCTACAGCACCTTTCCCGGCTTCGTGAGCTGGCGCGATCCCAAGTCCGGCTCTTGGTATGTGGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCATAGCGAGGACCTGCAGAGCCTGCTGCTGCGCGTGGCCAATGCCGTGAGCGTGAAGGGCATCTACAAGCAGATGCCAGGCTGCTTCAACTTCCTGCGGAAGAAGCTGTTCTTCAAGACCAGCGCCTC CTGA

In a second embodiment of this aspect of the invention there is provideda nucleic acid sequence encoding a chimeric protein having the formula:Ht1-L-Casp

whereinHt1 is a heterodimerization domain.L is an optional linker; andCasp is a caspase domain;

The nucleic acid sequence may encode the chimeric protein sequence shownas SEQ ID No. 2 or 3 or a variant thereof.

For example the nucleotide sequence may comprise the sequence shown asSEQ ID No. 13 or 14

(FKBP12-dCasp9) SEQ ID No. 13ATGCTGGAGGGCGTGCAGGTGGAGACCATCAGCCCAGGCGACGGCAGAACCTTCCCCAAGAGAGGCCAGACCTGCGTGGTGCACTATACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCCGCGACCGCAATAAGCCCTTCAAGTTCATGCTGGGCAAGCAGGAGGTGATCAGAGGCTGGGAGGAGGGCGTGGCCCAGATGAGCGTGGGCCAGAGAGCCAAGCTGACCATCAGCCCCGACTACGCCTATGGCGCCACCGGCCACCCCGGCATCATCCCACCCCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGAGTCCGGAGGCGGCTCCGGCGTGGATGGCTTCGGCGACGTGGGAGCCCTGGAGAGCCTGAGAGGCAACGCCGATCTGGCCTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCCGGGAGAGCGGCCTGCGGACCCGGACCGGCAGCAACATCGACTGCGAGAAGCTGAGGAGGCGCTTCTCCTCCCTGCACTTTATGGTGGAGGTGAAAGGCGATCTGACTGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCCCAGCAGGACCACGGAGCCCTGGATTGCTGTGTGGTGGTGATCCTGTCCCACGGCTGCCAGGCCAGCCACCTGCAGTTCCCCGGAGCCGTGTACGGCACCGACGGCTGTCCCGTGTCCGTGGAGAAGATCGTGAACATCTTCAACGGCACCTCCTGCCCCTCCCTGGGCGCCAAGCCCAAGCTGTTCTTTATCCAGGCCTGTGGCGGCGAGCAGAAGGACCACGGCTTTGAGGTGGCCAGCACCTCCCCCGAGGACGAGAGCCCAGGCAGCAACCCCGAGCCCGACGCCACCCCCTTCCAGGAGGGCCTGCGCACCTTCGACCAGCTGGACGCCATCAGCAGCCTGCCCACCCCCAGCGACATCTTCGTGAGCTACAGCACCTTTCCCGGCTTCGTGAGCTGGCGCGATCCCAAGTCCGGCTCTTGGTATGTGGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCATAGCGAGGACCTGCAGAGCCTGCTGCTGCGCGTGGCCAATGCCGTGAGCGTGAAGGGCATCTACAAGCAGATGCCAGGCTGCTTCAACTTCCTGCGGAAGAAGCTGTTCTTCAAGACCAGC GCCTCCTGA(FRB-dCasp9). SEQ ID No. 14ATGGCTTCTAGAATCCTCTGGCATGAGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCTCCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCTCAAAGCTCGAGTATAGCGGCGGCGGCAGCGGCGTGGATGGCTTCGGCGACGTGGGAGCCCTGGAGAGCCTGAGAGGCAACGCCGATCTGGCCTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCCGGGAGAGCGGCCTGCGGACCCGGACCGGCAGCAACATCGACTGCGAGAAGCTGAGGAGGCGCTTCTCCTCCCTGCACTTTATGGTGGAGGTGAAAGGCGATCTGACTGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCCCAGCAGGACCACGGAGCCCTCCATTCCTCTCTCCTCCTCATCCTCTCCCACCCCTCCCACCCCACCCACCTCCACTTCCCCCCACCCCTCTACCCCACCCACCCCTCTCCCCTCTCCCTCCACAACATCCTCAACATCTTCAACGGCACCTCCTGCCCCTCCCTGGGCGGCAAGCCCAAGCTGTTCTTTATCCAGGCCTGTGGCGGCGAGCAGAAGGACCACGGCTTTGAGGTGGCCAGCACCTCCCCCGAGGACGAGAGCCCAGGCAGCAACCCCGAGCCCGACGCCACCCCCTTCCAGGAGGGCCTGCGCACCTTCGACCAGCTGGACGCCATCAGCAGCCTGCCCACCCCCAGCGACATCTTCGTGAGCTACAGCACCTTTCCCGGCTTCGTGAGCTGGCGCGATCCCAAGTCCGGCTCTTGGTATGTGGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCATAGCGAGGACCTGCAGAGCCTGCTGCTGCGCGTGGCCAATGCCGTGAGCGTGAAGGGCATCTACAAGCAGATGCCAGGCTGCTTCAACTTCCTGCGGAAGAAGCTGTTCTTCAAGACCAGCGCCTCCTGA

In this second embodiment, the nucleic acid sequences may be provided inthe form of a construct which encodes both chimeric proteins.

The construct may encode a polyprotein having the formula:

Ht1-L2-Casp-coexpr-Ht2-L2-Casp

whereinHt1 is a first heterodimerization domain;L1 and L2 are optional linkers which may be the same or different;Coexpr is a sequence enabling coexpression of the two proteins:Ht1-L1-Casp and

Ht2-L2-Casp;

Ht2 is a second heterodimerization domain; andCasp is a caspase domain.

Where there are nucleic acid sequences encoding the same or similarsequences, such as the two caspase domains, one of the sequences may becodon wobbled to avoid homologous recombination.

In a third embodiment, nucleic acid sequence is provided which encodes asequence with the following formula:

Ht1-Casp-coexpr-Ht2-Ht2

whereinCasp is a caspase domain;Ht1 is a first heterodimerization domain;Coexpr is a sequence enabling coexpression of the proteins Ht1-Casp andHt2-Ht2, such as a cleavage site; andHt2 is a second heterodimerisation domain, which heterodimerises withHt1 in the presence of a chemical inducer of dimerization (CID).

In the sequence encoding the second protein, Ht2-Ht2, one of thesequences encoding Ht2 may be codon wobbled, in order to avoidhomologous recombination.

The nucleic acid construct according to the third embodiment may havethe sequence shown as SEQ ID No. 15.

(FKBP12-Casp9-2A-FRB-FRBw) SEQ ID No. 15ATGCTGGAGGGCGTGCAGGTGGAGACCATCAGCCCAGGCGACGGCAGAACCTTCCCCAAGAGAGGCCAGACCTGCGTGGTGCACTATACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCCGCGACCGCAATAAGCCCTTCAAGTTCATGCTGGGCAAGCAGGAGGTGATCAGAGGCTGGGAGGAGGGCGTGGCCCAGATGAGCGTGGGCCAGAGAGCCAAGCTGACCATCAGCCCCGACTACGCCTATGGCGCCACCGGCCACCCCGGCATCATCCCACCCCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGAGTCCGGAGGCGGCTCCGGCGTGGATGGCTTCGGCGACGTGGGAGCCCTGGAGAGCCTGAGAGGCAACGCCGATCTGGCCTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCCGGGAGAGCGGCCTGCGGACCCGGACCGGCAGCAACATCGACTGCGAGAAGCTGAGGAGGCGCTTCTCCTCCCTGCACTTTATGGTGGAGGTGAAAGGCGATCTGACTGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCCCAGCAGGACCACGGAGCCCTGGATTGCTGTGTGGTGGTGATCCTGTCCCACGGCTGCCAGGCCAGCCACCTGCAGTTCCCCGGAGCCGTGTACGGCACCGACGGCTGTCCCGTGTCCGTGGAGAAGATCGTGAACATCTTCAACGGCACCTCCTGCCCCTCCCTGGGCGGCAAGCCCAAGCTGTTCTTTATCCAGGCCTCTGCCGGCGAGCAGAAGGACCACGGCTTTGAGGTCGCCAGCACCTCCCCCGAGGACGAGAGCCCAGGCAGCAACCCCGAGCCCGACCCCACCCCCTTCCACCACCCCCTCCCCACCTTCCACCACCTCCACCCCATCACCACCCTCCCCACCCCCAGCGACATCTTCGTGAGCTACAGCACCTTTCCCGGCTTCGTGAGCTGGCGCGATCCCAAGTCCGGCTCTTGGTATGTGGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCATAGCGAGGACCTGCAGAGCCTGCTGCTGCGCGTGGCCAATGCCGTGAGCGTGAAGGGCATCTACAAGCAGATGCCAGGCTGCTTCAACTTCCTGCGGAAGAAGCTGTTCTTCAAGACCAGCGCCTCCCAGTGCACCAATTATGCTTTGCTTAAGCTGGCAGGCGATGTGGAATCAAACCCGGCTCCTGGGCTACAGGTCGAGACCATCTCTCCTGGCGACGGGAGAACATTTCCTAAAAGGGGCCAAACATGCGTGGTTCACTATACCGGTATGCTGGAGGATGGCAAAAAAGTAGACTCCAGCCGGGATAGAAACAAACCCTTTAAGTTCATGCTGGGTAAGCAGGAAGTTATACGGGGCTGGGAAGAGGGAGTAGCTCAGATGTCTGTGGGCCAGAGGGCCAAGCTGACCATCTCACCGGACTACGCCTACGGCGCTACCGGCCACCCTGGCATTATACCACCCCATGCAACTCTCGTCTTCGATGTTGAGTTGCTCAAACTGGAATCAGGCGGAGGCGGGTCTGGAGGAGGCGGCAGCATGCTGGAGGGCGTGCAGGTGGAGACCATCAGCCCAGGCGACGGCAGAACCTTCCCCAAGAGAGGCCAGACCTGCGTGGTGCACTATACCGCCATCCTGGAGGACCGCAAGAACTTCGACAGCAGCCGCGACCGCAATAAGCCCTTCAAGTTCATCCTGGCCAAGCAGGAGGTGATCAGAGGCTGGGAGGAGGGCGTGGCCCAGATGAGCGTGGGCCAGAGAGCCAAGCTGACCATCAGCCCCGACTACCCCTATCGCGCCACCGCCCACCCCCGCATCATCCCACCCCACCCCACCCTCGTGTTTGATGTGGAGCTGCTGAAGCTGGAG TCCTGA

Nucleic acid sequences with a high degree of similarity, such as thecaspase sequence(s) or FRB sequences may be codon wobbled to avoidrecombination.

Nucleic Acid Construct

The invention also provides a nucleic acid construct which comprises:

-   -   i) a first nucleic acid sequence encoding a chimeric protein        which comprises a caspase domain and a heterodimerization domain        which comprises an FK506-binding protein (FKBP); and    -   ii) a second nucleic acid sequence encoding a chimeric protein        which comprises a caspase domain and a heterodimerization domain        which comprises an FRB domain of mTOR.

The invention also provides a nucleic acid construct which comprises anucleic acid sequence encoding one or more chimeric protein(s) and afurther nucleic acid sequence of interest (NOI). The NOI may, forexample encode a T-cell receptor (TCR) or chimeric antigen receptor(CAR).

The nucleic acid sequences may be joined by a sequence allowingco-expression of the two or more nucleic acid sequences. For example,the construct may comprise an internal promoter, an internal ribosomeentry sequence (IRES) sequence or a sequence encoding a cleavage site.The cleavage site may be self-cleaving, such that when the polypeptideis produced, it is immediately cleaved into the discrete proteinswithout the need for any external cleavage activity.

Various self-cleaving sites are known, including the Foot-and-Mouthdisease virus (FMDV) 2a self-cleaving peptide, which has the sequenceshown as SEQ ID No. 16 or 17:

SEQ ID No. 16 RAEGRGSLLTCGDVEENPGP. or SEQ ID No 17 QCTNYALLKLAGDVESNPGP

The co-expressing sequence may be an internal ribosome entry sequence(IRES).

The co-expressing sequence may be an internal promoter.

T-Cell Receptor (TCR)

The T cell receptor or TCR is a molecule found on the surface of T cellsthat is responsible for recognizing antigens bound to majorhistocompatibility complex (MHC) molecules. The binding between TCR andantigen is of relatively low affinity and is degenerate: many TCRrecognize the same antigen and many antigens are recognized by the sameTCR.

The TCR is composed of two different protein chains, i.e. it is aheterodimer. In 95% of T cells, this consists of an alpha (α) and beta(β) chain, whereas in 5% of T cells this consists of gamma and delta(y/δ) chains. This ratio changes during ontogeny and in diseased states.

When the TCR engages with antigenic peptide and MHC (peptide/MHC), the Tlymphocyte is activated through a series of biochemical events mediatedby associated enzymes, co-receptors, specialized adaptor molecules, andactivated or released transcription factors.

The nucleic acid construct or vector of the present invention maycomprise a nucleic acid sequence encoding a TCR α chain, a TCR β chain,a TCRγ chain or a TCR δ chain. It may, for example, comprise a nucleicacid sequence encoding a TCR α chain and a nucleic acid sequenceencoding a TCR β chain; or a a nucleic acid sequence encoding a TCRγchain or a nucleic acid sequence encoding a TCR δ chain. The two nucleicacid sequences may be joined by a sequence enabling co-expression of thetwo TCR chains, such as an internal promoter, an IRES sequence or acleavage site such as a self-cleaving site.

Chimeric Antigen Receptors (CARs)

The nucleic acid sequence of interest (NOI) may encode a chimericantigen receptor (CAR).

Classical CARs are chimeric type I trans-membrane proteins which connectan extracellular antigen-recognizing domain (binder) to an intracellularsignalling domain (endodomain). The binder is typically a single-chainvariable fragment (scFv) derived from a monoclonal antibody (mAb), butit can be based on other formats which comprise an antigen binding sitesuch as a ligand. A spacer domain may be necessary to isolate the binderfrom the membrane and to allow it a suitable orientation. A commonspacer domain used is the Fc of IgG1. More compact spacers can sufficee.g. the stalk from CD8α and even just the IgG1 hinge alone, dependingon the antigen. A trans-membrane domain anchors the protein in the cellmembrane and connects the spacer to the endodomain which may comprise orassociate with an intracellular signalling domain.

Early CAR designs had intracellular signalling domains derived from theintracellular parts of either the y chain of the FcεR1 or CD3ζ.Consequently, these first generation receptors transmitted immunologicalsignal 1, which was sufficient to trigger T-cell killing of cognatetarget cells but failed to fully activate the T-cell to proliferate andsurvive. To overcome this limitation, compound signalling domains havebeen constructed: fusion of the intracellular part of a T-cellco-stimulatory molecule to that of CD3ζ results in second generationreceptors which can transmit an activating and co-stimulatory signalsimultaneously after antigen recognition. The co-stimulatory domain mostcommonly used is that of CD28. This supplies the most potentco-stimulatory signal—namely immunological signal 2, which triggersT-cell proliferation. Some receptors have also been described whichinclude TNF receptor family endodomains, such as the closely relatedOX40 and 41BB which transmit survival signals. Even more potent thirdgeneration CARs have now been described which have intracellularsignalling domains capable of transmitting activation, proliferation andsurvival signals.

CAR-encoding nucleic acids may be transferred to T cells using, forexample, retroviral vectors. In this way, a large number ofantigen-specific T cells can be generated for adoptive cell transfer.When the CAR binds the target-antigen, this results in the transmissionof an activating signal to the T-cell it is expressed on.

Thus the CAR directs the specificity and cytotoxicity of the T celltowards cells expressing the targeted antigen.

Vector

In a third aspect, the present invention provides a vector whichcomprises a nucleic acid sequence or nucleic acid construct of theinvention.

The present invention also provides a vector, or kit of vectors whichcomprises one or more nucleic acid sequence(s) or nucleic acidconstruct(s) of the invention and optionally one of more additionsnucleic acid sequences of interest (NOI). Such a vector may be used tointroduce the nucleic acid sequence(s) or nucleic acid construct(s) intoa host cell so that it expresses one or more chimeric protein(s)according to the first aspect of the invention and optionally one ormore other proteins of interest (P01). The kit may also comprise a CID.

The vector may, for example, be a plasmid or a viral vector, such as aretroviral vector or a lentiviral vector, or a transposon based vectoror synthetic mRNA.

The vector may be capable of transfecting or transducing a T cell.

The NOI may, for example encode a chimeric antigen receptor or a T-cellreceptor, such that when the vector is used to transduce a target cell,the target cell co-expresses a chimeric protein and a chimeric antigenreceptor or T-cell receptor.

Cell

The present invention also relates to a cell comprising a chimericprotein according to the first aspect of the invention.

The cell may express a chimeric protein having two heterodimerizationdomains, according of the first embodiment of the first aspect of thepresent invention.

The cell may express two chimeric proteins; one which comprises acaspase domain and a heterodimerization domain which comprises anFK506-binding protein (FKBP); and one which comprises a caspase domainand a heterodimerization domain which comprises an FRB domain of mTOR,according to the second embodiment of the first aspect of the invention.

There is also provided a cell which expresses two proteins:

-   -   Ht1-Casp and Ht2-Ht2        in which Ht1-Casp is a chimeric protein comprising a caspase        domain (Casp) and a first heterodimerization domain (Ht1); and        Ht2-Ht2 is an interfacing protein comprising two second        heterodimerization domains (Ht2)        such that, in the presence of a chemical inducer of dimerization        (CID), a pair of the chimeric proteins Ht1-Casp9 interact such        that Ht1 from each chimeric protein heterodimerizes with an Ht2        domain from the interfacing protein, causing homodimerization of        the two caspase domains (see FIG. 1d ).

The cell may, for example, be an immune cell such as a T-cell or anatural killer (NK) cell.

The cell may be a stem cell such as a haematopoietic stem cell.

T cells or T lymphocytes which are a type of lymphocyte that play acentral role in cell-mediated immunity. They can be distinguished fromother lymphocytes, such as B cells and natural killer cells (NK cells),by the presence of a T-cell receptor (TCR) on the cell surface. Thereare various types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells inimmunologic processes, including maturation of B cells into plasma cellsand memory B cells, and activation of cytotoxic T cells and macrophages.TH cells express CD4 on their surface. TH cells become activated whenthey are presented with peptide antigens by MHC class II molecules onthe surface of antigen presenting cells (APCs). These cells candifferentiate into one of several subtypes, including TH1, TH2, TH3,TH17, Th9, or TFH, which secrete different cytokines to facilitatedifferent types of immune responses.

Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells andtumor cells, and are also implicated in transplant rejection. CTLsexpress the CD8 at their surface. These cells recognize their targets bybinding to antigen associated with MHC class I, which is present on thesurface of all nucleated cells. Through IL-10, adenosine and othermolecules secreted by regulatory T cells, the CD8+ cells can beinactivated to an anergic state, which prevent autoimmune diseases suchas experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+ or CD8+. Memory T cellstypically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells,are crucial for the maintenance of immunological tolerance. Their majorrole is to shut down T cell-mediated immunity toward the end of animmune reaction and to suppress auto-reactive T cells that escaped theprocess of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells.

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Tregcells) arise in the thymus and have been linked to interactions betweendeveloping T cells with both myeloid (CD11c+) and plasmacytoid (CD123+)dendritic cells that have been activated with TSLP. Naturally occurringTreg cells can be distinguished from other T cells by the presence of anintracellular molecule called FoxP3. Mutations of the FOXP3 gene canprevent regulatory T cell development, causing the fatal autoimmunedisease IPEX.

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originateduring a normal immune response.

Natural Killer Cells (or NK cells) are a type of cytolytic cell whichform part of the innate immune system. NK cells provide rapid responsesto innate signals from virally infected cells in an MHC independentmanner

NK cells (belonging to the group of innate lymphoid cells) are definedas large granular lymphocytes (LGL) and constitute the third kind ofcells differentiated from the common lymphoid progenitor generating Band T lymphocytes. NK cells are known to differentiate and mature in thebone marrow, lymph node, spleen, tonsils and thymus where they thenenter into the circulation.

Stem cells are undifferentiated cells which can differentiate intospecialized cells. In mammals, there are two broad types of stem cells:embryonic stem cells, which are isolated from the inner cell mass ofblastocysts, and adult stem cells, which are found in various tissues.In adult organisms, stem cells and progenitor cells act as a repairsystem for the body, replenishing adult tissues. In a developing embryo,stem cells can differentiate into all the specialized cells—ectoderm,endoderm and mesoderm (see induced pluripotent stem cells)—but alsomaintain the normal turnover of regenerative organs, such as blood,skin, or intestinal tissues.

There are three known accessible sources of autologous adult stem cellsin humans:

1. Bone marrow, which requires extraction by harvesting, i.e. drillinginto bone.2. Adipose tissue, which requires extraction by liposuction.3. Blood, which requires extraction through apheresis, wherein blood isdrawn from the donor and passed through a machine that extracts the stemcells and returns other portions of the blood to the donor.

Adult stem cells are frequently used in medical therapies, for examplein bone marrow transplantation. Stem cells can now be artificially grownand transformed (differentiated) into specialized cell types withcharacteristics consistent with cells of various tissues such as musclesor nerves. Embryonic cell lines and autologous embryonic stem cellsgenerated through Somatic-cell nuclear transfer or dedifferentiation canalso be used to generate specialised cell types for cell therapy.

Hematopoietic stem cells (HSCs) are the blood cells that give rise toall the other blood cells and are derived from mesoderm. They arelocated in the red bone marrow, which is contained in the core of mostbones.

They give rise to the myeloid (monocytes and macrophages, neutrophils,basophils, eosinophils, erythrocytes, megakaryocytes/platelets,dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).The hematopoietic tissue contains cells with long-term and short-termregeneration capacities and committed multipotent, oligopotent, andunipotent progenitors.

HSCs are a heterogeneous population. Three classes of stem cells exist,distinguished by their ratio of lymphoid to myeloid progeny (L/M) inblood. Myeloid-biased (My-bi) HSC have low L/M ratio (between 0 and 3),whereas lymphoid-biased

(Ly-bi) HSC show a large ratio (>10). The third category consists of thebalanced (Bala) HSC, whose L/M ratio is between 3 and 10. Only themyeloid-biased and balanced HSCs have durable self-renewal properties.

The chimeric protein-expressing cells of the invention may be any of thecell types mentioned above.

T or NK cells expressing one or more chimeric protein(s) according tothe first aspect of the invention may either be created ex vivo eitherfrom a patient's own peripheral blood (1^(st) party), or in the settingof a haematopoietic stem cell transplant from donor peripheral blood(2^(nd) party), or peripheral blood from an unconnected donor (3^(rd)party).

Alternatively, T or NK cells expressing one or more chimeric protein(s)according to the first aspect of the invention may be derived from exvivo differentiation of inducible progenitor cells or embryonicprogenitor cells to T cells. Alternatively, an immortalized T-cell linewhich retains its lytic function and could act as a therapeutic may beused.

In all these embodiments, chimeric protein(s)-expressing cells aregenerated by introducing DNA or RNA coding for the, or each, chimericprotein, and optionally an NOI by means such as transduction with aviral vector or transfection with DNA or RNA.

The cell of the invention may be an ex vivo T or NK cell from a subject.The T or NK cell may be from a peripheral blood mononuclear cell (PBMC)sample. T or NK cells may be activated and/or expanded prior to beingtransduced with nucleic acid encoding one or more chimeric protein(s)according to the first aspect of the invention, for example by treatmentwith an anti-CD3 monoclonal antibody.

The T or NK cell of the invention may be made by:

-   -   (i) isolation of a T or NK cell-containing sample from a subject        or other sources listed above; and    -   (ii) transduction or transfection of the T or NK cells with one        or more a nucleic acid sequence(s) according to the second        aspect of the invention.

The present invention also provides a kit which comprises a T or NK cellcomprising one or more chimeric protein(s) according to the first aspectof the invention and a CID.

Pharmaceutical Composition

The present invention also relates to a pharmaceutical compositioncontaining a plurality of cells according to the fourth aspect of theinvention. The pharmaceutical composition may additionally comprise apharmaceutically acceptable carrier, diluent or excipient. Thepharmaceutical composition may optionally comprise one or more furtherpharmaceutically active polypeptides and/or compounds. Such aformulation may, for example, be in a form suitable for intravenousinfusion.

Methods

The invention also provides a method for making a cell according to thefourth aspect of the invention which comprises the step of transducingor transfecting a cell with a vector according to the third aspect ofthe invention.

The vector may, for example, be a retroviral or lentiviral vector.

The invention also provides a method for deleting a cell according tothe fourth aspect of the invention, which comprises the step of exposingthe cells to the CID, such as rapamycin or a rapamycin analog. The cellsmay be exposed to the CID in vivo or in vitro. Deletion of the cell maybe caused by apoptosis induced by caspase activation, followingCID-induced homodimerization of the caspase domains.

The CID may be administered in the form of a pharmaceutical composition.The pharmaceutical composition may additionally comprise apharmaceutically acceptable carrier, diluent or excipient. Thepharmaceutical composition may optionally comprise one or more furtherpharmaceutically active polypeptides and/or compounds. Such aformulation may, for example, be in a form suitable for intravenousinfusion.

The invention also provides a method for preventing and/or treating anpathological immune reaction in a subject caused by administration of acell according to the fourth aspect of the invention to the subject,which comprises the step of administering a CID, such as rapamycin or arapamycin analog to the subject.

The pathological immune reaction may be selected from the followinggroup: graft-versus-host disease; on-target, off-tumour toxicity; immuneactivation syndrome; and lymphoproliferative disorders.

The invention also provides a method for treating or preventing adisease in a subject, which comprises the step of administering a cellaccording to the fourth aspect of the invention to the subject. The cellmay be in the form of a pharmaceutical composition as defined above.

The method may comprises the following steps:

-   -   (i) transducing or transfecting a sample of cells isolated from        a subject with a vector according to the third aspect of the        invention, and    -   (ii) administering the transduced/transfected cells to a        patient.

A method for treating a disease relates to the therapeutic use of thecells of the present invention. Herein the cells may be administered toa subject having an existing disease or condition in order to lessen,reduce or improve at least one symptom associated with the diseaseand/or to slow down, reduce or block the progression of the disease.

The method for preventing a disease relates to the prophylactic use ofthe immune cells of the present invention. Herein such cells may beadministered to a subject who has not yet contracted the disease and/orwho is not showing any symptoms of the disease to prevent or impair thecause of the disease or to reduce or prevent development of at least onesymptom associated with the disease. The subject may have apredisposition for, or be thought to be at risk of developing, thedisease.

The methods for treating a disease provided by the present invention mayinvolve monitoring the progression of the disease and monitoring anytoxic activity and adjusting the dose of the CID administered to thesubject to provide acceptable levels of disease progression and toxicactivity.

Monitoring the progression of the disease means to assess the symptomsassociated with the disease over time to determine if they arereducing/improving or increasing/worsening.

Toxic activities relate to adverse effects caused by the cells of theinvention following their administration to a subject. Toxic activitiesmay include, for example, immunological toxicity, biliary toxicity andrespiratory distress syndrome.

In particular the invention provides a method for treating a disease ina subject, which comprises the following steps:

(i) administering a cell according to the fourth aspect of the inventionto the subject;(ii) monitoring the subject for the development of a pathological immunereaction; and(iii) administering rapamycin or a rapamycin analogue to the subject ifthe subject shows signs of developing or having developed a pathologicalimmune reaction.

The present invention provides a cell of the present invention for usein treating and/or preventing a disease.

The cell may, for example, be for use in haematopoietic stem celltransplantation, lymphocyte infusion or adoptive cell transfer.

The invention also relates to the use of a cell of the present inventionin the manufacture of a medicament for the treatment and/or preventionof a disease.

The present invention also provides a CID agent capable inducingdimerizing a chimeric protein according to the first aspect of theinvention for use in treating and/or preventing a toxic activity.

The present invention also provides a CID agent for use in activating apair of caspase domains of chimeric proteins according to the firstaspect of the invention in a cell.

The disease to be treated and/or prevented by the cells and methods ofthe present invention may be an infection, such as a viral infection.

The methods of the invention may also be for the control of pathogenicimmune responses, for example in autoimmune diseases, allergies andgraft-vs-host rejection.

Where the cells of the invention express a TCR or CAR, they may beuseful for the treatment of a cancerous disease, such as bladder cancer,breast cancer, colon cancer, endometrial cancer, kidney cancer (renalcell), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreaticcancer, prostate cancer and thyroid cancer.

The TCR/CAR-expressing cells of the present invention may be capable ofkilling target cells, such as cancer cells.

The invention also provides rapamycin or a rapamycin analogue for use inpreventing or treating a pathological immune reaction caused byadministration of a cell according to the fourth aspect of the inventionto a subject.

The cells of the present invention may be used in any cellular therapyin which modified or unmodified cells are administered to a patient. Anexample of a cellular therapy is adoptive T cell transfer after CD34+stem cell transplantation. Administering T cells after stem celltransfer helps to accelerate the reconstitution of an immune system inthe patient recipient. When a matched related or unrelated donor is notavailable, or the disease is too aggressive for an extensive donorsearch, the use of an HLA haploidentical family donor may be effective.Such donors may be parents, siblings, or second-degree relatives. Suchinfusions may enhance immune recovery and thereby reduce virusinfections and eliminate relapsing leukemia cells. However, thecoexistence of alloreactive T cells in a donor stem cell graft may causegraft-versus-host disease (GvHD) in which the donor cells react againstthe recipient, which may progressively damage the skin, gut, liver, andother organs of the recipient.

Other examples of cell therapies include using native cells or cellsgenetically engineered to express a heterologous gene. These treatmentsare used for many disorders, including blood disorders, but thesetherapies may have negative side effects. In another method, immatureprogenitor cells that can differentiate into many types of mature cells,such as, for example, mesenchymal stromal cells, may be used to treatdisorders by replacing the function of diseased cells. There presentinvention provides a rapid and effective mechanism to remove possiblenegative effects of donor cells used in cellular therapy.

The present invention provides a method of reducing the effect of graftversus host disease in a human patient following donor T celltransplantation, comprising transfecting or transducing human donor Tcells in a donor cell culture with vector according to the presentinvention; administering the transduced or transfected donor

T cells to the patient; subsequently detecting the presence or absenceof graft versus host disease in the patient; and administering achemical inducer of dimerization (CID) to a patient for whom thepresence of graft versus host disease is detected. The T cells may benon-allodepleted.

The present invention provides a method of stem cell transplantation,comprising administering a haploidentical stem cell transplant to ahuman patient; and administering haploidentical donor T cells to thepatient, wherein the T cells are transfected or transduced in ahaploidentical donor cell culture with a vector according to theinvention.

The cells may be non-allodepleted human donor T cells in a donor cellculture.

The present invention also provides a method of stem celltransplantation, comprising administering a haploidentical stem celltransplant to a human patient; and administering non-allodepletedhaploidentical donor T cells to the patient, wherein the T cells aretransfected or transduced in a haploidentical donor cell culture withvector according to the invention.

The haploidentical stem cell transplant may be a CD34+ haploididenticalstem cell transplant. The human donor T cells may be haploidentical tothe patient's T cells. The patient may any disease or disorder which maybe alleviated by stem cell transplantation. The patient may have cancer,such as a solid tumour or cancer of the blood or bone marrow. Thepatient may have a blood or bone marrow disease. The patient may havesickle cell anemia or metachromatic leukodystrophy. The donor cellculture may be prepared from a bone marrow sample or from peripheralblood. The donor cell culture may be prepared from donor peripheralblood mononuclear cells. In some embodiments, the donor T cells areallodepleted from the donor cell culture before transfection ortransduction. Transduced or transfected T cells may be cultured in thepresence of IL-2 before administration to the patient.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1—Production of T-cells Expressing Chimeric Proteins

T-cells were transduced with the different constructs. For thetwo-molecule rapCasp9 (FIG. 1a ), T-cells were transduced with twovectors: one coding for FKBP12-Casp9 co-expressed with the greenfluorescent protein eGFP by means of an internal ribosome entrysequence, and the other coding for FRB-Casp9 co-expressed with the bluefluorescent protein eBFP2. For the one molecule rapCasp9 (FIG. 1b ),T-cells were transduced with just one vector coding for the respectiverapCasp9 which are co-expressed eGFP. A construct which providedFKB12-Casp9 and FRB-FRBw was encoded in a tri-cistronic cassette wherebythe FKBP12-Casp9 and FRB-FRBw were co-expressed using a FMD-2A likepeptide and eGFP was co-expressed with an IRES. The T-cells wereintentionally only partially transduced so within the cell culture aproportion of cells remained non-transduced to act as an internalnegative control. As a further control, T-cells were transduced with avector which codes for eGFP alone to exclude non-specific effects ofRapamycin on transduced cells.

Example 2—Testing Deletion of Chimeric Protein-Expressing Cells withRapamycin

T-cells were exposed to different concentrations of Rapamycin andincubated for 48 hours. Following this, T-cells were stained withAnnexin-V and 7AAD and analysed by flow-cytometry. By gating on the livecells, and interrogating the population of cells expressing fluorescentproteins, survival of the transduced and non-transduced populationscould be clearly measured. The dual FRB-Casp9 and FKBP12-Casp9 approachresulted in effective deletion of only double positive cells asexpected. The FKBP12-FRB-Casp9 construct resulted in effective deletionof single positive cells. The FKBP12-Casp9-FRB construct resulted inminimal deletion. The FKBP12-Casp9/FRB-FRBw resulted in effectivedeletion of single positive cells. The control resulted in no specificdeletion (FIGS. 2 and 3).

Example 3—Testing an Expanded Set of Constructs

The constructs shown in FIG. 5 we generated and transduced into Jurkatcells. Transduced cells were mixed with non-transduced (NT) cells tohave both construct positive and negative cells within the population.Rapamycin was added at a concentration of 0, 1, 10, 100 and 1000 nM andthe cells were incubated for 24 h.

Following harvesting, the cells were stained with PI and annexin V andanalysed by FACS. The results are shown in FIGS. 6 to 9 and summarisedin FIG. 10.

The construct which has a configuration as defined according to thefirst embodiment of the first aspect of the invention, namely MP20244,performed very well in this assay, giving very efficient killing oftransfected cells at all concentrations of rapamycin above and including1 nM.

The pair of constructs having a configuration as defined according tothe second embodiment of the first aspect of the invention, namelyMP20206 and MP20207 also performed very well, giving very efficientkilling of transfected cells at all concentrations of rapamycin aboveand including 1 nM.

The construct having a configuration as defined according to the thirdembodiment of the first aspect of the invention, namely MP20265, alsoperformed well, giving some killing at 1 nM rapamycin and efficientkilling at concentrations of rapamycin of 10 nM and above.

Constructs having a configuration as defined according to the fourthembodiment of the first aspect of the invention, namely MP20263, MP20264and MP21067 prefomed well at 1 nM rapamycin, but at higherconcentrations of rapamycin killing was less efficient.

Example 4—Testing the Constructs with Temsirolimus

In an equivalent experiment to the one described in Example 3, cellsexpressing the constructs shown in FIG. 5 were treated with bothrapamycin and temsirolimus, a rapamycin analogue.

As with the experiment outlined in Example 3, the transduced Jurkatcells were mixed with non-transduced (NT) giving a population containingboth cells expressing the constructs and non-transduced cells.

Cells at a concentration of with 2×10⁵ cells per well were either leftuntreated, or were treated with rapamycin or temsirolimus at thefollowing concentrations: 0.01, 0.1, 1, 10 nM (of either rapamycin ortemsirolimus)

Cells were incubated for 24 h and were then stained for Annexin V and PIand were analysed by FACS. The results are shown in FIG. 11.

An equivalent pattern of Jurkat cell killing was observed with thevarious constructs shown in FIG. 5 in the presence of temsirolimus ashad been previously observed in the presence of rapamycin.

In particular, the construct MP20244, which has a configuration asdefined according to the first embodiment of the first aspect of theinvention; and the pair of constructs MP20206 and MP20207, having aconfiguration as defined according to the second embodiment of the firstaspect of the invention, both performed well. Both gave efficientkilling of transfected cells at all concentrations of temsirolimus aboveand including 1nM.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments.

Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. A chimeric protein having the formula:Ht1 -Ht2-Casp wherein Casp is a caspase domain; Ht1 is a firstheterodimerization domain; and Ht2 is a second heterodimerization domainand wherein, in the presence of a chemical inducer of dimerization(CID), an identical pair of the chimeric proteins interact such that Ht1from one chimeric protein heterodimerizes with Ht2 from the otherchimeric protein, causing homodimerization of the two caspase domains.2. A chimeric protein according to claim 1 wherein Ht1 does notheterodimerize with Ht2 within the same chimeric protein.
 3. A chimericprotein according to claim 1, wherein the caspase domain comprises aninitiator caspase selected from the following group: caspase-8,caspase-9 and caspase-10.
 4. A chimeric protein according to claim 1,wherein the caspase domain comprises an executioner caspase selectedfrom caspase-3 and caspase-7.
 5. A chimeric protein according to claim 1wherein one heterodimerization domain comprises an FK506-binding protein(FKBP) and the other heterodimerization domain comprises an FRB domainof mTOR.
 6. A chimeric protein according to claim 5, wherein Ht1comprises FRB and Ht2 comprises FKBP.
 7. A chimeric protein according toclaim 5, wherein the CID is rapamycin or a rapamycin analog. 8.(canceled)
 9. A nucleic acid sequence which encodes a chimeric proteinaccording to claim
 1. 10. A nucleic acid construct which comprises oneor more nucleic acid sequence(s) according to claim 9 and a nucleic acidsequence encoding a T-cell receptor (TCR) or chimeric antigen receptor(CAR).
 11. (canceled)
 12. A nucleic acid construct having the structure:Ht1-Casp-coexpr-Ht2-Ht2 wherein: Casp is a nucleic acid sequenceencoding a caspase domain; Ht1 is a nucleic acid sequence encoding afirst heterodimerization domain; Ht2 is a nucleic acid sequence encodinga second heterodimerization domain; and coexpr is a nucleic acidsequence allowing co-expression of Ht1-Casp and Ht2-Ht2, whereinexpression of the nucleic acid construct results in the production of achimeric protein Ht1-Casp and an interfacing protein Ht2-Ht2 andwherein, in the presence of a chemical inducer of dimerization (CID), apair of the chimeric proteins Ht1-Casp9 interact such that Ht1 from eachchimeric protein heterodimerizes with an Ht2 domain from the interfacingprotein, causing homodimerization of the two caspase domains.
 13. Anucleic acid construct according to claim 12, wherein Ht1 comprises anFK506-binding protein (FKBP) and Ht2 comprises an FRB domain of mTOR.14. A nucleic acid construct according to claim 11, which also comprisesa nucleic acid sequence encoding a T-cell receptor (TCR) or chimericantigen receptor (CAR).
 15. (canceled)
 16. A vector which comprises anucleic acid sequence according to claim 9 which also comprises anucleotide of interest.
 17. A vector according to claim 16, wherein thenucleotide of interest encodes a chimeric antigen receptor or a T-cellreceptor, such that when the vector is used to transduce a target cell,the target cell co-expresses a chimeric protein according claim 1 and achimeric antigen receptor or T-cell receptor.
 18. A cell which expressesa chimeric protein according to claim
 1. 19. (canceled)
 20. A cell whichexpresses two proteins: Ht1-Casp and Ht2-Ht2 wherein Ht1-Casp is achimeric protein comprising a caspase domain (Casp) and a firstheterodimerization domain (Ht1); and Ht2-Ht2 is an interfacing proteincomprising two second heterodimerization domains (Ht2) wherein, in thepresence of a chemical inducer of dimerization (CID), a pair of thechimeric proteins Ht1-Casp9 interact such that Ht1 from each chimericprotein heterodimerizes with an Ht2 domain from the interfacing protein,causing homodimerization of the two caspase domains. 21.-25. (canceled)26. A method for preventing or treating a disease in a subject, whichcomprises the step of administering to the subject, (i) a cell whichexpresses a chimeric protein having the formula:Ht1-Ht2-Casp wherein Casp is a caspase domain, Ht1 is a firstheterodimerization domain, and Ht2 is a second heterodimerization domainand wherein, in the presence of a chemical inducer of dimerization(CID), an identical pair of the chimeric proteins interact such that Ht1from one chimeric protein heterodimerizes with Ht2 from the otherchimeric protein, causing homodimerization of the two caspase domains;or (ii) a cell which expresses two proteins: Ht1-Casp and Ht2-Ht2wherein Ht1-Casp is a chimeric protein comprising a caspase domain(Casp) and a first heterodimerization domain (Ht1) and Ht2-Ht2 is aninterfacing protein comprising two second heterodimerization domains(Ht2) wherein, in the presence of a chemical inducer of dimerization(CID), a pair of the chimeric proteins Ht1-Casp9 interact such that Ht1from each chimeric protein heterodimerizes with an Ht2 domain from theinterfacing protein, causing homodimerization of the two caspasedomains.
 27. A method according to claim 26: wherein the celladministered to the subject is a cell that was isolated from the subjectand transduced or transfected with a vector encoding the protein orproteins.
 28. A method according to claim 27 for treating cancer. 29.The method of claim 26, which further comprises the step ofadministering rapamycin or a rapamycin analog to the subject if thesubject shows signs of developing or having developed a pathologicalimmune reaction caused by the administration of the cell.
 30. A methodaccording to claim 29, wherein the pathological immune reaction isselected from the following group: graft-versus-host disease; on-target,off-tumour toxicity; immune activation syndrome; and lymphoproliferativedisorders. 31-33. (canceled)